Celiac Vagus Nerve Mediates Monocytic α7nAchR Expression In The Spleen:Implications For Inflammation Modulation

The vagal-spleen axis is an important pathway in regulating inflammatory response,and is associated with developing inflammatory diseases,and improving therapeutics.Studies has veridlied that the spleen is required for the vagal cholinergic anti-inflammatory activity to maintain systemic immune homeostasis,but the underlying mechanism of this function is not fully understood yet.Although cholinergic anti-inflammatory pathway is centered on systemic cytokine modulation by alpha 7 nicotinic acetylcholine receptor(α7n ACh R)activation of spleen-residue macrophage,this mechanism is unable to fully explain inflammation regulation in distal tissues,suggesting that other mechanism might exist.We hypothesized that vagus nerve mediates α7n ACh R expression in monocytes,and the spleen is essential site for this process.This investigation was designed to test this possibility.To evaluate the effect of the spleen and vagus nerve on α7n ACh R expression in circulating monocytes,mice were subjected to splenectomy or sham operation,and celiac vagotomy or sham operation.Therefore,mice were divided into four groups: splenectomy group,sham group,vagotomy group,and sham group.After surgery for 7 days,blood was collected via the retro-orbital venous plexus from each group of mice and circulating monocytes were isolated,the level of α7n ACh R expression in circulating monocytes was analyzed by Western blot(WB).To determine the mechanism underlying vagus nerve regulating monocytic α7n ACh R expression in the spleen,we detected nerve-related neuregulin-1(NRG-1)expression in the spleen by reverse transcription-polymerase chain reaction(RT-PCR)in vagotomized mice,and observed in vitro the effect of NRG-1on α7n ACh R expression in monocytes from vagotomized mice.To evaluate the pathway that NRG-1 acts in monoyctes,we analyzed the expression of NRG-1 recepotr epidermal growth factor-receptor family of tyrosine kinases(Er BB)in monocytes.Subsequently,these monocytes were pre-treated with Erb B4-blocking antibody prior to NRG-1,and the effect of NRG-1 on α7n ACh R expression in monocytes was detected by RT-PCR.To know the effect of the loss of moocytic α7n ACh R expression on inflammatory status in peripheral multiple tissues,vagotomized mice were intraperitoneally injected with lipopolysaccharide(LPS,a well-established experimental tool to study inflammatory response in vitro and in vivo)and nicotine(an effective agonist of α7n ACh R),and the levels of pro-inflammatory cytokine tumor necrosis factor(TNF-α)and interleukin-1beta(IL-1β)were measured by ELISA.We found that splenectomy or coeliac vagotomy abrogated α7n ACh R expression in circulating monocytes.Western blot showed that a specific antibody anti-α7n ACh R recognized a clear band with 55 k D in circulating monocytes from sham operation mice,but did not detect the expression ofα7n ACh R in the monocytes from splenectomized or vagotomized mice.RT-PCR data showed that NRG-1 m RNA level decreased significantly in the spleen of vagotomized mice as compared with sham mice,suggesting potential role of NRG-1 in modulating α7n ACh R expression in monocytes.NRG-1 treatment in vitro can induce α7n ACh R m RNA expression in cultured monocytes from vagotomized mice.RT-PCR analysis showed that the level of expression of Erb B2 and Erb B3 was below the threshold of detection,and Erb B4 m RNA significantly expresses.After Er BB4 was blocked by the antibody,NRG-1 treatment fails to trigger α7n ACh R m RNA expression in monocytes.Moreover,After celiacl vagotomy for 7 days,LPS challenge significantly increase in the levels of TNF-α and IL-1β in plasma,liver,kidney and skeletal muscle from vagotomized mice as compared with sham mice,and nicotine administration did not attenuate these cytokine levels in these tissues from vagotomized mice,suggesting that vagus nerve-mediated α7n ACh R expression in monocytes in the spleen contributes to inflammation modulation in peripheral multiple tiuuses.Our results reveal that celiac vagus nerve mediates α7n ACh R expression in monocytes through NRG-1/Erb B4 pathway via ERK signaling pathway of MAPK,and the spleen is indispensable site for this process.